1. Field of the Invention
The present invention is related to a protein modified to improve its stability and a method for producing the same. More particularly, the present invention is related to a protein coupled to xcex2-1,3-glucan branched with xcex2-1,6-linkage, which shows an improved stability while retaining its activity, to a method for the production thereof, and to a composition for external or topical application comprising the stabilized proteins.
2. Description of the Related Arts
Proteins play various roles in living body, for example as an biocatalyst in the metabolisms, as signal transmitting agents, or the like and are commonly used advantageously in a variety of applications such as laundry industry (e.g. detergents), cosmetic industry, pharmaceutical compositions (e.g. digestants, anti-inflammatory drugs and the like), food industry (e.g. meat tenderizing agents) and so on.
However, their uses have been limited due to their instability. antigenicity, or other safety problems. In fact, in addition to skin irritation, unsatisfactory effectiveness on the skin and/or difficulty in the production of the external or topical application compositions such as cosmetic or dermatological compositions incorporated with the proteins, the short shelf-life has restricted the practical use of these compositions.
Several attempts such as immobilization or chemical modification have been made to improve the stability of the proteins so far. Although these attempts did not provide a satisfactory result, representative examples thereof are explained below.
U.S. Pat. No. 4,556,554 teaches a cosmetic composition for removal of sebum exudate from the skin, which comprises immobilized enzymes in cosmetically acceptable vehicles, and the enzymes are immobilized to functional polymers in known manners by chemical and or physical means. They are released from immobilization upon application to the skin. However, this immobilization does not provide a satisfactory improvement of stability of the enzymes in the composition.
Masunaga et al (T. Masunaga et al., IFSCC, Yokohama, A205, pp 483-501) reports proteases which are chemically coupled to polyethylene glycol. The proteases show improved stability and reduced skin-irritation. But they still have drawbacks that their shelf-time is not sufficiently prolonged and their preparation is complex.
U.S. Pat. No. 5,230,891 assigned to Kanebo Limited teaches a modified protease and their production method. Polysaccharide such as dextran, alginic acids or carrageenin is reacted with cyanuric trichloride to give a triazine ring-bound polysaccharide, which is reacted with protease to give a protease coupled to polysaccharide via triazine ring. This method still has a drawback that it is very complicated.
EP 0,803,257 A2 and JP 4-141097A teach an enzyme stabilization by oxidative process using polysaccharides. Thus-stabilized enzymes can, for example, be attached to surfaces of medical devices to minimize undesirable biological reaction associated with medical devices or attached to the surface of the reactor. However, it is not certain the stabilized enzymes can be incorporated into cosmetic or dermatological compositions
Therefore, there has been a need to provide proteins so stabilized so as to be incorporated into compositions for external or topical applications.
The present invention provides a modified protein, which has an improved stability, said protein being coupled to xcex2-1,6 residue branched from xcex2-1,3-glucan.
The present invention further provides a method for producing the modified protein, which comprises the steps of
(a) reacting a glucan with periodates to oxidize xcex2-1,6 residue of the glucan into aldehyde;
(b) removing unreacted periodates from the reaction solution;
(c) adding to the reaction solution a protein in an amount of 0.00001xcx9c10.0% by weight and allowing the protein to contact with the oxidized glucan; and
(d) adding to the reaction solution a reducing agent in an amount of 0.0001xcx9c1.0% by weight.
The method may further comprise the step of washing the product obtained in the step (d).
The present invention still provides a composition for external or topical application, which comprises the modified protein as an active ingredient.
The present invention will be described in detail below.
According to the present invention, the protein is stabilized by coupling to xcex2-1,3-glucan regularly branched with xcex2-1,6-linkage (herein after referred to simply as xe2x80x9cglucanxe2x80x9d). The glucan has a neutral pH in aqueous phase, has a triple helix shape contributing to its stability, and can be modified only at its xcex2-1,6-residue by chemical means. In contrast, polysaccharides such as dextran are modified randomly or at every residue. Therefore, glucan allows a unique modification of proteins in terms of coupling index between glucan and protein, and stability and activity of the modified protein.
The glucan employed according to the invention may include schizophyllan originated from Schizopyllum commune, scleroglucan from Sclerotinia sp., lentinan from Lentinus edodes. Other glucans can be employed as long as they have xcex2-1,3-chain branched with regular xcex2-1,6-linkage.
The glucans as naturally occurred have usually a molecular weight ranging from several hundred thousands to several millions. They can be used as they are, or treated by mechanical or chemical means to have a molecular weight from about 50,000 to several hundred thousands. For example, microfluidizer. sonicator or xcex2-glucanase treatment can be used to break the intact glucan to give a smaller molecular weight glucans. The term of xe2x80x9cglucanxe2x80x9d is used herein to include the intact glucans as well as the small molecular weight glucans.
The glucans may be used in an aqueous solution having a concentration of 0.001xcx9c20.2% by weight, and preferably 0.1xcx9c5.0% by weight.
The proteins which can be modified to improve their stability according to the present invention may include, but not limited thereto, proteases such as papain, bromelain, ficin, trypsin, chymotrypsin, collagenase, elastase, plasmin or bacterial protease; carbohydrate-lysis enzymes such as amylase, glucoamylase, cellulase, pectinase, xylanase, xcex1-glucosidase, xcex2-glucosidase, xcex2-galactosidase, xcex2-galactosidase, xcex2-glucanase, chitinase or mannanase; lipases such as phospholipase or triacylglycerol hydrolase; nucleic acid lysis enzymes such as DNase or RNase; phosphatases; lysozymes; catalases; superoxide dismutases; transglutamninases, peroxidases; photolyases; complex enzymes originated from microorganisms; cytokines; growth factors; hormones; antigens; antibodies; immunoglobulins; lactoferrins; metallothioneins; thioredoxins; antimicrobial proteins; or antioxidative proteins;. The proteins also include active peptides thereof or conjugates with saccharides or lipids. They may be used singly or in combinations thereof.
The method for the production of the modified proteins will be described in detail hereinafter.
The method comprises the steps of (a) reacting a glucan with periodates to oxidize xcex2-1,6-linkage of the glucan into aldehyde; (b) removing unreacted periodates from the reaction solution; (c) adding to the reaction solution a protein in an amount of 0.00001xcx9c10.0% by weight and allowing the protein to contact with the oxidized glucan; and (d) adding to the reaction solution a reducing agent in an amount of 0.0001xcx9c1.0% by weight. The method may further comprise the step of washing the desired product obtained in the step (d).
The step (a) is to oxidize xcex2-1,6-residue of the glucan into aldehyde, which allows a coupling between the glucan and the protein. Periodates are used in an amount of 0.1xcx9c50.0 molar equivalents, and preferably 2xcx9c5 molar equivalents with respect to one mole of glucan. The reaction mixture is allowed to react in the dark with stirring. Periodates may include, but not limited to, HIO4, H5O6, NaIO4, Na3H2IO4 or KIO4. After the reaction, ethylene glycol may be added to the reaction mixture to stop the excessive oxidation reaction, if necessary. The ethylene glycol may be used in an amount of 0.01xcx9c10.0% by weight.
The step (b) is to remove the unreacted periodates and may be carried out in known manners such as dialysis or ultrafiltration using a membrane having a molecular cut off of 1,00xcx9c500,000 and preferably 10,000xcx9c100,000.
The step (c) is to react the oxidized glucan in the step (a) and the protein to form Schiff""s base between the aldehyde of xcex2-1,6-residue of the glucan and amino group of the protein. The protein is used in an amount of 0.00001xcx9c10.0% by weight and preferably 0.001xcx9c5.0% by weight based on the glucan solution obtained in the step (b).
The step (d) is reduce the Schiff""s base and may be carried out by adding a reducing agent in an amount of 0.001xcx9c1.0% by weight and preferably 0.01xcx9c0.1% by weight based on the Schiff""s base solution in the step (c). The reducing agent may include, but not limited to, sodium borohydride(NaBH4), sodium cyanoborohydride (NaBH4), amine borane or the like. At this step, lysine may be added in an amount of 0.01xcx9c10% by weight and preferably 0.1xcx9c1.0% by weight to the reaction mixture to neutralize the unreacted aldehyde group.
After the step (d), the modified (stabilized) protein obtained in the step (d) may be washed by known methods such as dialysis or ultrafiltration using a membrane having a molecular cut off of 1,000xcx9c500,000 and preferably 10,000xcx9c100,000 (Step (e)).
All the steps is preferably carried out under conditions which do not cause any degeneration or activity loss of the protein to be modified. Such conditions may include, but not limited to, the temperature of about 0xc2x0 C. to room temperature with mild stirring.
For the present invention, the different proteins may be modified simultaneously or in turn.
The stabilized protein may be incorporated into cosmetic or dermatological compositions for topical or external application for use in corneum-removal, sebum control, anti-inflammation, skin soothing, acne control, anti-oxidation, toxin clearance, metal ion clearance, skin elasticity improvement, wrinkle removal, anti-aging, skin whitening, tanning, depilatory prevention and inhibition, hair-removal, anti-bacterial or anti-fungal action, deodorization, sun-burn repair, wound healing and the like. The amount of the stabilized protein to be incorporated in the composition may be decided by the ordinary skilled in the art and may be in a range from 0.00001 to 100% by weight. Thus, the compositions for external or topical application according to the present invention comprises 0.00001 to 100% by weight of the stabilized protein and cosmetically or dermatologically acceptable vehicles. These vehicles are well known to the skilled in the art and may be chosen depending on the kind, formulation and usage of the composition.
The composition may have a cosmetic formulation such as cleansing lotion, cleansing cream, skin milk, emollient lotion, massage cream, emollient cream, make-up base, lipstick, facial pack, facial gel, shampoos, rinses, hair-tonics, or soaps, or dermatological composition such as lotions, ointments, gels, creams, patches or sprays.
The present invention will be described by way of various preparation and experimental examples, but should not be interpreted to be limited to these examples.